If baseline and follow-up echocardiograms were compared, there was a similar but significant improvement in both EF and GLS in both separate study groups. An immediate invasive strategy in NSTE-ACS patients did not result in an improved left ventricular EF or GLS recovery compared with an early strategy.The timing of coronary angiography in patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) remains a matter of debate. The relationship between the timing of invasive management and left ventricular function (LVF) is largely unknown. The An Immediate or Early Invasive Strategy in Non-ST-Elevation Acute Coronary Syndrome trial (OPTIMA-2) was a randomized controlled prospective open-label multicenter trial that randomized 249 NSTE-ACS patients to either an immediate (<3 h) invasive treatment strategy or an early strategy (12–24 h). Patients were pre-treated with a combination of aspirin, ticagrelor and fondaparinux. The aim of this prespecified sub-analysis was to assess (the recovery of) left ventricular function by analysing echocardiography data obtained <72 h after admission and at 30-day follow-up, for patients with a confirmed diagnosis of acute coronary syndrome. LVF was determined using ejection fraction (EF) and global longitudinal strain (GLS). Inter-observer variability was tested. No difference in the recovery of EF was found between an immediate and early strategy if the follow-up echocardiograms were compared to baseline: 2.5% (standard deviation (SD): 7.9) and 3.3% (SD: 8.5),  For non-ST-segment elevation acute coronary syndromes (NSTE-ACS) a routine invasive strategy is advised according to both the European Society of Cardiology (ESC) and American College of Cardiology (ACC)/American Heart Association (AHA) guidelines ,2. In adThe “An Immediate or Early Invasive Strategy in Non-ST-Elevation Acute Coronary Syndrome trial” (OPTIMA-2) was designed to study the impact of the timing of an invasive strategy on the total infarct size combining both the effects of spontaneous myocardial infarction and periprocedural-related injury in patients presenting with NSTE-ACS, with the use of modern antiplatelet and anticoagulant therapy. The main results of the OPTIMA-2 have been published previously. In short, no significant difference in infarct size (measured as area under the curve (AUC) of creatine kinase myocardial band (CKMB)) or clinical endpoints  was found between an immediate (<3 h) and early (12–24 h) invasive strategy .Echocardiography is the most-used imaging modality to assess cardiac function in the acute phase of the NSTE-ACS. In addition, global longitudinal strain (GLS) measurement with speckle tracking echocardiography represents an emerging modality for more accurate assessment of  cardiac function ,9. GLS hThe OPTIMA-2 trial was a prospective, open-label, randomized controlled trial  performed by the OLVG hospital in Amsterdam, the Netherlands. The complete study design and the main clinical outcomes have been published previously . In shorAfter enrolment, all patients were given ticagrelor and aspirin (loading dose) directly, except those patients already using aspirin and/or a P2Y12 inhibitor at the time of admission. Fondaparinux was administered to all patients without contra-indication.All patients underwent cardiac ultrasound within 72 h after admission (baseline), and a follow-up echocardiogram was performed after 30-days (follow-up). The method of revascularization after initial angiography (percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG)) was at the discretion of the treating physician or the consensus of the hospital’s heart team. The operator was allowed to use iFR, IVUS or OCT if necessary or to decide to perform ad-hoc PCI after index angiography, but this was not mandatory for the study. For this sub-study of the OPTIMA-2 trial, we analysed only the echocardiographic data of patients with a confirmed diagnosis of NSTE-ACS.The echocardiographic examinations were performed by experienced sonographers on General Electric (GE) Vivid S6 or Vivid E9 machines . The images were analysed offline with General Electric EchoPac (version 112) software by an experienced investigator blinded to clinical baseline data and endpoints. Left ventricular ejection fraction (LVEF) was calculated using Simpson’s biplane method obtained from apical four- and two chamber views.Speckle tracking 2D analysis was performed in the apical 4-chamber, 2-chamber and the longitudinal long-axis  views. The endocardium of the LV was traced semi automatically and adjusted manually in case the investigator deemed the tracings to be inaccurate. Segmentation was performed according to the AHA 17-segment model. Each apical view covered six segments. The width of the tracings covered the endo-, myo- and epicardium and was adjusted per segment if necessary. GLS was calculated as the mean value of the peak systolic longitudinal strain of each segment at a frame rate of 40–90 frames/second.There were two primary endpoints: (1) The comparison between an immediate and an early invasive strategy in improvement of LVEF of the 30-day follow-up echocardiogram in comparison to baseline . (2) The comparison between an immediate and an early invasive strategy in the improvement of GLS if the 30-day follow-up echocardiogram was compared to baseline.Secondary endpoints were differences in LVEF or GLS measurements at baseline and at 30-day follow-up, if both study groups were compared at each specific time point. In addition, for each individual study group separately, the improvement in LVEF or GLS measurement was calculated if the 30-day follow-up was compared to the baseline echocardiogram.Follow-up consisted of a visit to an outpatient’s clinic and follow-up echocardiography after 30 days of being discharged. If it was not possible to contact a patient, his or her vital status was obtained from the local authorities.t-test, paired t-test  or one-way ANOVA test. In order to assess the correlation between different variables and changes in left ventricular function (LVF), we first analysed the data using univariate linear regression analysis for the following variables: above 60 years of age, diabetes mellitus, hypertension, current smoking, hypercholesterolemia, peripheral arterial disease, family history of ischemic heart disease, several biomarkers  and high-sensitive C-reactive protein (hs-CRP), duration of chest pain before admittance, syntax score, number of diseased coronary arteries, treatment strategy , target vessel, total numbers of stents implanted during index procedure and Global Registry of Acute Coronary Events (GRACE)-risk score at index admission. Beta coefficients were calculated with 95% confidence interval (CI)  . In caseAnalysis of EF and GLS was done by a single blinded observer. A second observer blindly analysed 20 randomly selected patients and a total of 40 echocardiograms, at the same heartbeat as the first observer. Subsequently, inter-observer reliability was tested by the calculation of the intraclass correlation coefficient (ICC). A two-way mixed model of the absolute agreement type was used. ICC values greater than 0.9 were considered excellent reliability .Between March 2013 and November 2018, a total of 249 patients were included in the OPTIMA-2 study. In total, 125 patients were randomized to the immediate invasive group and 124 to the early invasive group. In total, 15 (12%) patients in the immediate group and 12 (10%) patients in the early group ultimately did not have a final diagnosis of NSTE-ACS and were therefore excluded from this analysis . The basIn total, 173 (77.8%) baseline echocardiograms were eligible for analysis of EF and 174 (78.4%) GLS. Follow-up echocardiograms were obtained after a median of 31 days (IQR 27–33), after which another 186  EF and 183 (82.4%) GLS measurements were included. The patients that were unsuitable for EF calculation due to a lack of echo quality were in most cases also unsuitable for GLS calculation due to the same reason (90% overlap of patients at baseline and 95% at follow-up). The echocardiographic excluding process is shown in Flow chart of available echocardiography data of participating patients that were randomized to either an immediate invasive strategy or an early invasive strategy.p = 0.51  7.9 vs. 3.3%, SD 8.5, p = 0.51 . In addip = 0.82 .If baseline data was compared, we found no difference in LVEF between the study groups, nor did we find any difference between the groups at 30-day follow-up LVEF .p < 0.001) and the early invasive group (p < 0.001) separately. In addition, GLS improved significantly if the 30-day follow-up echocardiogram was compared to baseline in the immediate invasive group (p < 0.001) and the early invasive group (p < 0.001).When GLS was compared we found similar results: no differences between the study groups at baseline or at 30-day follow-up echocardiogram . The LVEBy univariate analyses, only NT-proBNP (admission) was statistically significantly associated with change in EF or GLS. The change in EF or GLS for the sub-group of patients treated by PCI were also compared and results are presented in the p = 0.29), nor in improvement in GLS at 30-day follow-up (p = 0.21). For the sub-group of patients treated by PCI, a comparison between the different randomization groups was made regarding improvement in LVEF or GLS. No differences between the immediate (n = 43) and early (n = 54) invasive groups were observed in LVEF: 2.1% (SD 9.1) vs. 4.6% (SD 8.8), p = 0.15) or GLS: −0.8% (SD 2.6) vs. −1.1% (SD 2.7), p = 0.55.If the different methods of treatment  were compared, we found no significant difference in LVEF improvement at 30-day follow-up  for the LVEF measurements and 0.91 (95% CI: 0.81–0.95) for the GLS measurements.The main findings of the present analysis are: (1) There was no difference in improvement in LV function between patients randomized to an immediate (<3 h) or an early (12–24 h) invasive strategy, if 30-day follow up echocardiogram was compared to baseline. (2) LV function improved significantly in both groups with respect to EF and GLS recovery, if baseline was compared to 30-day follow-up echocardiogram.Several trials have compared LVEF in NSTE-ACS patients according to (the timing of) treatment strategy. However, these trials are of heterogenic methodology, often not powered to detect a difference in LV function and did not use GLS to assess LV function. Tekin et al. compared an early invasive strategy (<24 h) to a delayed invasive strategy group (24–72 h) with respect to echocardiographic LVEF with a 3-month echocardiographic follow-up. A total of 131 NSTEMI patients participated in the study. Infarct-related arteries were equally divided between the study groups and the majority of patients in both groups had three-vessel disease. After 3 months, the prevalence of recurring MI, cardiac related hospitalization and death was significantly lower in the early invasive strategy group. While the discharge values of LVEF were similar (56.5% and 55.6%) between the two treatment strategies, the EF after 3 months in the early invasive group improved (59.3%) significantly more compared to the delayed invasive group (54.1%) .To the best of our knowledge, there are no studies comparing GLS outcomes for different timing strategies in NSTE-ACS patients. In line with our GLS results there is a study published by Grenne et al. that showed the deterioration of GLS in NSTE-ACS patients awaiting angiography. A total of 56 patients with NSTEMI were allocated to an early (<32 h) invasive strategy. Patients planned for very early (<10 h) strategy were excluded. Ultrasound GLS measurements were performed after admission and before angiography. The GLS values deteriorated significantly (from −16.1% ± 2.6% to −15.0% ± 2.6%) awaiting angiography, implying the progressive impairment of myocardial function by delaying an invasive strategy. The authors suggested benefit from immediate revascularization to relieve ischemia and prevent myocardial damage. However, after 3 months, follow-up GLS improved significantly compared to normal values (mean GLS −17.1% ± 3.1%) [A plausible explanation for the improvement of EF and GLS function in both study groups in our current study is the effect of coronary revascularization itself and medical therapy allowing the recovery of myocardial stunning . StrikinSeveral limitations should be considered with respect to the current study. First, the current data is collected in a randomized controlled setting. However, the original study was powered to show a difference in AUC of CKMB. For this reason and the fact that the study was terminated early, it was not specifically powered to show a difference in EF and GLS. Second, several factors in the study design and baseline characteristics are limitations to the current sub-analysis: The study was single-center and had a long period of patient recruitment for a relatively small and heterogeneous study population. In addition, an outside core laboratory for analysis was not used. The reduction of EF at the initial hospitalization was mild for most patients. Patients were mostly male with low SYNTAX scores, and about 66% of patients had positive troponins. Third, 20% of echocardiographic data was not eligible for EF and/or GLS analysis. This was in most cases due to poor image quality, often in the semi-acute setting. It is not uncommon that part of the echocardiographic data is not suitable for analysis; lack of sufficient image quality to perform GLS in a non-acute clinical setting is reported in 7% of cases . As our In NSTE-ACS patients, if a direct (<3 h) invasive strategy was compared to an early (12–24 h) strategy, it did not result in a difference in LVEF or GLS recovery after 30 days. However, at 30-day follow-up, the LVF in both study groups showed a significant improvement in comparison to baseline echocardiography.


Objectives: To study the effect of the optical zone diameter of ablation on higher order aberrations after transepithelial photorefractive keratectomy for myopia and myopic astigmatism.Methods: In this historical cohort study in 2019, patients were grouped into 7-mm (Gr-1) and 6.5-mm optical zones of ablation (Gr-2). Topographic and higher order aberrations at analysis diameters of 2, 4, and 6 mm were measured before and six months after transepithelial photorefractive keratectomy. The changes in the five types of higher order aberrations in the 6.5 mm and 7 mm groups were compared. The pupillary diameter was correlated with the change in the higher order aberrations.Results: We had 24 eyes of 12 patients in Gr-1 and 80 eyes of 40 patients in Gr-2. The trefoil type of higher order aberrations at 6 mm was significantly more prevalent in Gr-2 than in Gr-1 before surgery (p = 0.038). The change in spherical aberration six months after surgery compared with before was significantly more at 6 mm in the eyes of Gr-2 patients (p = 0.02). For the eyes managed by the 7-mm optical zone of ablation for transepithelial photorefractive keratectomy, the decline in the different types of higher order aberrations was significant. The pupillary diameter was positively correlated with the change in the third-order coma in Gr-2 . All the eyes had an uncorrected visual acuity of 0.0 LogMAR in Gr-1 and 95% in Gr-2 after surgery.Conclusions: The higher order aberrations six months after transepithelial photorefractive keratectomy were similar in eyes managed with 7-mm and 6.5-mm optical zone for ablation. But a lower aberration coefficient in eyes was managed by the 7-mm zone than the 6.5-mm zone of optical ablation at 6-mm analysis diameter. The human population is facing a myopia epidemic . A numbeSeveral studies have reviewed the impact of different types of refractive surgeries on HOAs and the role of the optical zone diameter for ablation in causing discomfort due to a change in HOAs. These surgeries include femtosecond laser-assisted surgery, laser in situ keratomileusis, laser-assisted sub-epithelial keratectomy (LASEK), photorefractive keratectomy, and Epi-LASIK (laser-assisted in situ keratomileusis) ,6,7-10.The effect of the optical zone diameter selected to perform photorefractive keratectomy excimer laser treatment for myopia has been examined, finding that the 7 mm optical zone provides better outcomes when treating some HOAs . Some reMyopic patients in the Middle East undergo refractive surgery since the climatic conditions are often not conducive for contact lenses. A dry humid climate also poses challenges to patients’ decision to undergo refractive surgeries as tear film abnormalities also can cause HOAs .In this study, we present the comparison of 6.5-mm and 7-mm diameter optical zones on the change in different types of HOAs following T-PRK among patients with myopia and myopic astigmatism and its determinants.Our study is a retrospective, consecutive, and non-randomized cohort study. It included eyes operated by single-step T-PRK from September to December 2019 at a private eye clinic in the Kingdom of Saudi Arabia. The local research and ethics committee approved this study. The tenets of the Helsinki declaration for human research were strictly followed. The patient was adequately informed about the study as well as the risks and benefits of the surgery, and signed informed consent was obtained.We tested the hypothesis that HOAs are significantly less in T-PRK surgeries performed using the 7-mm optical zone than the 6.5-mm optical zone for ablation . To achiThe inclusion criteria included age above 18 years, primary myopia or compound myopic astigmatism, preoperative manifest refraction spherical equivalent (MRSE) within the range of −1.50 to −7 D, a stable refraction for at least one year before the surgery, contact lens discontinuation for at least three weeks, and an estimated residual stromal corneal bed thickness of >350 μm at the thinnest location. The exclusion criteria included previous ocular surgery, active ocular diseases, corneal dystrophy, retinal disease, dry eye, a history of severe eye trauma, irregular astigmatism or suspected keratoconus on corneal topography, any previous ocular surgery, glaucoma or glaucoma suspect, diabetes mellitus, autoimmune diseases, pregnancy, breastfeeding, and moderate-to-severe dry eye.Preoperative information included age, sex, and the eye operated. Patients were instructed not to wear contact lenses for at least 14 days to 21 days before the measurements were to be taken. The uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA) were noted for distance using Snellen’s visual acuity chart projected at six meters from the patient. The corneal topography was evaluated using a Pentacam camera , and tomography was carried out using Sirius . We used an aberrometer to measure the wavefront aberration to detect the third-order coma value, third-order trefoil value, fourth-order spherical aberration value, aberration coefficient, and Q value. The measurement of each eye included keratometry K1, K2, central corneal thickness, and pupillary diameter in normal daytime illumination in a room. Cycloplegic refraction was performed, and then spherical, cylindrical, and spherical equivalent refractive power in a diopter was noted for each eye.The ablations were performed using an Amaris 500 Hz excimer laser . The machine was kept in aberration-free mode. The treatment goal was to maintain emmetropia.Before the surgery, moxifloxacin 0.5%  drops were applied. An antiseptic chlorhexidine gluconate 0.05% solution  was used to clean the eyelids before surgery. Eyes were kept open during surgery using a wire lid speculum.The application of the laser and other surgical steps are described in detail in the literature ,17. The All the measurements relating to distance visual acuity, topography, and HOA were repeated six months after surgery using the same equipment by the same investigators. For each HOA, the values noted six months after surgery were compared with those noted before surgery to determine the change in HOA.The data were collected using a pretested data collection form. They were collected from both records and a printout of the equipment software files. In cases of disputes, the data in the equipment linked to the case record number were considered correct.The data were transferred into a Statistical Package for the Social Sciences (SPSS v25)  spreadsheet. The qualitative variables were presented as numbers and percentages. The quantitative variables were plotted to study the distribution. If the distribution was normal, we presented the mean and standard deviation for each group. If the distribution was skewed, we presented their median and interquartile range. To compare the findings of the two groups, we used univariate analysis to estimate the differences in the means, 95% confidence intervals, and two-sided P values based on a matched-pair analysis. To compare the outcomes of the two groups that were not normally distributed, we performed a nonparametric analysis using two related sample calculations and presented the two-sided Wilcoxon P test values. A p-value of <0.05 was considered statistically significant.Our study comprised 24 eyes of 12 patients  managed using the 7-mm optical zone for ablation and 80 eyes of 40 patients  managed by the 6.5-mm optical zone of ablation. The mean age of patients in Gr-1 and Gr-2 were 22.9 ± 2.9 years and 25.1 ± 4.1 years, respectively (p = 0.08). There were seven (58.3%) men in Gr-1 and 12 (27.9%) men in Gr-2 (p = 0.009). Nine (37.5%) right eyes in Gr-1 and 43 (53.8%) in Gr-2 were operated (p = 0.17). The HOAs before and after six months T-PRK in 6-mm diameter of analysis of a patient is shown in Figure The comparison of the topographic parameters and refractive status of the eyes in both groups before T-PRK is given in Table They were not significantly different. The UCVA of the eyes in Gr-1 before surgery was 0.22 ± 0.21 LogMAR and that of the eyes in Gr-2 was 0.24 ± 0.2 LogMAR (p = 0.69). The BCVA of the eyes in Gr-1 was 0.0 LogMAR and that of the eyes in Gr-2 was 0.0 LogMAR.The presence of different types of HOAs before T-PRK at 2, 4, and 6 mm diameters from the center of the cornea was compared in Gr-1 and Gr-2 . In Gr-1, the mean ablation zone was 7.95 ± 1.86 mm, and in Gr-2, it was 7.6 ± 0.9 mm (p = 0.38). The comparison of HOAs noted six months after T-PRK in Gr-1 and Gr-2 was not statistically different .The correlation of the pupillary diameter and change in the HOA values were correlated in Gr-1 and Gr-2 Table .Apart from the third-order coma type of HOA at 4 mm in the 6.5-mm optical ablation zone , no other HOAs in either group had a significant correlation with the change in HOA due to T-PRK.All the eyes managed by the 7-mm optical zone for ablation for T-PRK had a UCVA of 0.0 LogMAR at six months. Of the 80 eyes managed by the 6.5-mm optical zone of ablation for T-PRK, 76 (95%) eyes had a UCVA of 0.0 LogMAR and the remaining four (5%) had a UCVA of 0.1 LogMAR.HOA was noted in all the eyes treated with T-PRK. All the eyes showed a correction of LOAs but still had HOAs to some extent six months after T-PRK. At that time, the HOAs were no different in the eyes treated with the 7-mm and 6.5-mm optical zones of T-PRK. The increase in the fourth-order spherical aberration, however, was significantly more at the 6-mm diameter of analysis if the 6.5-mm optical zone was selected for T-PRK. The pupillary diameter was significantly and positively correlated with the change in the third-order coma in the eyes treated with T-PRK using the 6.5-mm optical zone ablation.This is perhaps the first study describing the differences between the 6.5-mm and 7-mm optical zones for T-PRK ablation while addressing the HOAs among myopic and myopic astigmatic patients. The selection of a wider optical zone does not make any significant difference when changes in HOAs are desired to compensate for preexisting HOAs. The present study also confirms the need to include wavefront technology to improve vision both for the LOAs and for the HOAs as well as to minimize iatrogenic HOAs in refractive surgery .In the present study, we noted two outcomes: (1) A difference in the HOA values at six months in the 7-mm and 6.5-mm optical zone groups subjected to T-PRK and (2) a change in the HOA values at six months compared with those before T-PRK in these two groups. We did not find any significant difference in the HOA values in the 7-mm and 6.5-mm optical zone groups at six months after T-PRK. By contrast, Ozulken et al. noted significantly fewer spherical aberrations and lower aberration coefficient values in the 7-mm group than those in the 6.5-mm group . The groThe comparison of pre- and post-HOA values is crucial as T-PRK is now also used to address existing HOAs and LOAs in eyes with refractive errors ,18. ThusAddressing the existing HOAs is an accepted norm in refractive surgeries to improve the quality of life, especially reducing the issues of glare, halos, and night vision . HoweverA number of refractive surgeries are practiced to provide spectacle-free vision. However, to address both LOA and HOAs, wavefront-guided T-PRK has been noted to be more effective and safer than femtosecond LASIK.  For bothCompared with previous studies, larger optical zones are now used for ablation with modern tools. Seo et al. compared the outcomes of 6-mm and 6.5-mm ablation zones in 2004, while our study compared 7 mm with 6.5 mm and found advantages and disadvantages in relation to addressing HOAs .Single-step T-PRK is recommended to address LOAs effectively in mild to severe myopic eyes . HoweverIn our study, the HOAs were more in eyes that underwent the 6.5-mm optical zone of ablation instead of the 7-mm optical ablation zone, and the pupillary diameter affected this correlation. Night driving and contrast sensitivity are known complaints in patients after refractive surgeries, even if LOA is satisfactorily corrected. HOAs in eyes with a disparity in the optical zone of ablation and pupillary diameter could extenuate these problems in a mesopic setting ,24. ThisIn the present study, we compared the impact of selecting smaller optical zones of ablation on changes in HOAs after T-PRK. It would be interesting to study long-term changes through a longer follow-up; there are different refractive surgeries performed by different equipment. A study to review the impact of the tools used for refractive surgery on the HOA would also be of interest.There were a few limitations in our study. The data are from one institution and managed by one surgeon. Therefore, operator’s variability could pose a challenge while extrapolating the study outcomes to larger populations. The outcomes were based on a six-month follow-up after T-PRK. Corneal healing could take longer. Hence, studies with long-term efficiency and safety are recommended.T-PRK is efficient in addressing LOAs. The selection of the optical zone diameter for ablation in T-PRK was not beneficial in addressing HOAs. As such, there is no significant difference in HOAs six months after T-PRK in those managed with the 7-mm and 6.5-mm optical zones, but a decline in HOAs compared to before surgery was noted; especially, the fourth-order spherical aberration in the 6.5-mm group was more than that in the 7-mm group of T-PRK at 6-mm analysis diameter. The pupillary diameter has an independent influence on the changes in the third-order coma type of HOA six months after T-PRK.


SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the virus passes from the upper respiratory tract to the myocardium. We hypothesized that SARS-CoV-2 is present in the blood of COVID-19 infected patients, spreading to other organs such as heart.We targeted two viroporins, Orf3a and E, in SARS-CoV-2. Orf3a and E form non-voltage-gated ion channels. A combined fluorescence potassium ion assay with three channel modulators  was developed to detect SARS-CoV-2 Orf3a/E channel activity. In blood samples, we subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity.In lentivirus-spiked samples, we detected significant channel activity of Orf3a/E based on increase in fluorescence induced by 4-aminopyridine, and this increase in fluorescence was inhibited by emodin and gliclazide. In 18 antigen/PCR-positive samples, our test results found 15 are positive, demonstrating 83.3% concordance. In 24 antigen/PCR-negative samples, our test results found 21 are negative, showing 87.5% concordance.We developed a cell-free test that can detect Orf3a/E channel activity of SARS-CoV-2 in blood samples from COVID-19-infected individuals, confirming a hypothesis that the virus spreads to the heart via blood circulation. COVID-19 infection is caused by the SARS-CoV-2 virus. Various testing methods are used to detect SARS-CoV-2 viral RNA, viral antigen, and antibodies formed in response to COVID-19 infection or vaccination -10. COVIRNA has been detected by RT-PCR in up to 76% of blood or serum samples collected from patients with COVID-19 infection [viral proteins have not been reported in blood samples of COVID-19 patients by three testing methods.SARS-CoV-2 In addition to primary infection in the respiratory tract, SARS-CoV-2 can cause damage in multiple organs including the heart. The initial hypothesis presumed that virus-mediated inflammation was the primary cause of cardiac dysfunctions ,15. ReceSARS-CoV-2 contains two viroporins, Orf3a and E, both of which can form non-voltage-gated cation channels ,25,26. WUse of blood samples from COVID-19 patients was approved by the West Virginia University Institutional Review Board. Research was discussed with each patient, and if the patient consented to the research project, whole blood was drawn by standard venipuncture into one tube, containing EDTA for anticoagulation. Plasma/sera samples were stored in tube without EDTA. A total of 42 samples were collected: 28 whole blood samples and 14 plasma/serum samples.+) to permeate K+ channels [+ channels are open, Tl+ in the extracellular solution flows down its concentration gradient into the cells via K+ channels. Inside the cells, Tl+ binds to and activates a fluorogenic indicator dye preloaded into the cells, resulting in a dramatic increase in fluorescence signal. This technique allows rapid determination of K+ channel activity in a high-throughput manner [A fluorescence-based potassium ion channel assay utilizes the ability of thallium  were purchased from Sigma. Stock solution (50 mm) was prepared in DMSO. Approximately 0.5 mm emodin and gliclazide were used in ∼100 uL solution. DMSO at ∼1–2% of test solution had no effects on test results.http://n2t.net/addgene:141383-LV; RRID:Addgene_141383) [http://n2t.net/addgene:141385-LV; RRID:Addgene_141385) [6 TU/mL, containing ∼1000 functional lentiviral particles in the experiments (TU: transduction unit).Lentivirus containing SARS-CoV-2 Orf3a and lentivirus containing E were obtained from Addgene donated by Nevan Krogen lab: pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro was a gift from Nevan Krogan  . pLVX-EF_141385) . We usedAntigen testing was performed in the clinical laboratory using the CareStart Rapid Diagnostic Test for the Detection of SARS-CoV-2 Antigen . Briefly, the nasopharyngeal swab is removed from pouch and the swab is introduced to the nasal passage until it reaches the posterior nasopharynx. The swab is rotated 3–5 times over the posterior nasopharynx and then removed from the nostril with rotation to sample the anterior nares. In the laboratory, the seal is removed from the extraction vial containing the extraction buffer. The swab is placed in the extraction vial and rotated vigorously 5 times. The extraction vial is then squeezed while the swab is removed by rotating against the sides of the extraction vial to remove any excess fluid from the swab, and a cap is placed on the extraction vial. The sample is mixed by tapping the bottom of the extraction tube, inverted, and 3 drops are squeezed into the sample well. Results are read at ten minutes.A red control line will appear at top of well by the letter “C.” If the test is positive, a blue line will appear below the red line and across from the letter “T.” Positive result reveals red and blue lines. Negative results reveal only a single red line. A result with a blue line without a red line is invalid and test is repeated.Fluorescence data were collected using Gen5 2.0 microplate reader software (BioTek), processed in Excel, and analyzed and plotted using GraphPad Prism 8.Table Figure + permeability [Figure eability  and are Figure In lentivirus containing E, 4-AP (1 mM) induced an increase in fluorescence  and specificity (97%) , servingRecently, a mass-spectrometry-based high-throughput test that targeted proteomics (targeting nuvleoprotein peptides) from “nasopharyngeal and oropharyngeal swabs” was reported . CombineIn this exploratory research, we targeted Orf3a and E channels for several reasons. First, Orf3a is present only in SARS-CoV-1 and SARS-CoV-2; therefore, a combination of Orf3a and E channel activity is highly selective. Second, highly sensitive detection methods based on fluorescence are used to detect channel activity. Third, utilization of Orf3a and E channel blockers allows us to detect signals only from Orf3a/E while eliminating background fluorescence in blood by subtracting the fluorescence signals in samples without channel blockers. Fourth, protein sequence motifs that control channel activity of Orf3a/E are highly conserved. Thus, detecting the conserved channel activity of Orf3a/E may serve as a valuable tool as viral variants proliferate worldwide. Other interpretations of these preliminary findings are possible.The sample size is not large in this work, and samples were collected when disease prevalence was high (approximately up to 50%) in the point-of-care clinic. A larger sample size is needed to test our method for detecting functional viral proteins in blood at lower disease prevalence.Our new testing method for SARS-CoV-2 may complement present testing by identifying functional virus in blood samples in a 96-well plate with a total experimental time of around 2 hours. The method can be easily expanded to 384-well plate, and the experimental time can be shortened to less than 1 hour with automation. In addition, our test targets conserved motifs in the Orf3a/E proteins, which are not affected by virus variants. This focus on virally conserved components could alleviate concerns regarding the accuracy of molecular-based methodologies as new variants spread through the community. Finally, detection of the SARS-CoV-2 in blood provides a reasonable explanation as to how the virus spreads from the point of entry in the respiratory tract to other organs including the heart.


Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR . We also found that disease activity was positively correlated with HuR expression ( Immune-mediated necrotizing myopathy (IMNM) is an autoimmune disease that is characterized by severe muscle weakness and myofiber necrosis, but usually low inflammatory cell infiltration and lesser involvement of other organs . IMNM coMicroRNAs (miRNA) participate in skeletal muscle differentiation . miR-17-RNA-binding proteins (RBPs) are known to change the fate or function of bound RNA. Of such RBPs, HuR, a member of the ELAVL1/Hu family, has been reported to modulate the fusion of myoblasts into myotubes during skeletal myogenesis by coordinating the expression of myogenic regulatory factors such as myogenin (MyoG), MyoD, and p21 –16. NeveIn this study, we aimed to explore the role and mechanism of abnormally expressed miR-18a-3p and its target RBP HuR in the myogenesis of patients with IMNM. Our results may help in better understanding the pathogenesis of IMNM and developing effective therapeutic targets.Patients with IMNM, DM and healthy controls (HCs), who participated in this study, were admitted to the Department of Rheumatology and Physical Examination from 2017 to 2019 in China–Japan Friendship Hospital, Beijing, China. The age and gender of the patients with IMNM were matched with those of the HCs. IMNM was classified according to the pathological criteria described at the 224th European Neuromuscular Center International Workshop . A 10 cm visual analog scale (VAS) was used to evaluate the muscle activity component of the myositis disease activity assessment tool (MDAAT-muscle) and physician global assessment (PGA) for disease activity .Serum anti-HMGCR antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) kit . Serum anti-SRP antibodies were detected using a commercially available kit .2. The cells were seeded in 12-well or 6-well plates and incubated for 18 h before transfection. At 80–90% confluence, the cells were transferred into Dulbecco’s modified Eagle medium supplemented with 2% horse serum and 1% penicillin-streptomycin for inducing differentiation.Human skeletal muscle myoblasts (HSMM) were obtained from Lonza Bioscience  and cultured using the Clonetics skeletal muscle myoblast cell system (Lonza) in a humidified incubator  at 37°C with 5% COHSMM were seeded in 12-well or 6-well plates; when the cells reached approximately 70% confluence, they were transfected with 100 nM of small interfering RNA targeting HuR (siHuR) or small interfering control RNA (siCtrl), miR-18a-3p mimics or mimics negative control (NC) and miR-18a-3p inhibitor or inhibitor NC  using Lipofectamine 3000  according to the manufacturer’s instructions. The culture system was replaced with fresh medium at 12 h post-transfection. The cells were used to stimulate differentiation at 24 h post-transfection.Dual-luciferase reporter gene assay was used to confirm the binding sites of miR-18a-3p and the 3′ untranslated region (UTR) of HuR mRNA. A pmirGLO-HuR-wild type (Hur-WT) or a pmirG-LO-HuR-mutant type (HuR-MUT) and miR-18a-3p mimics or mimics NC were co-transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). Dual-Glo luciferase assay system  was used to measure the luciferase activity in the cell lysates according to the manufacturer’s instructions. The ratios of Firefly to Renilla luciferase activities were used as the result of the dual-luciferase reporter gene assay. The experiments were performed in three independent replicates.http://mirwalk.umm.uni-heidelberg.de/) and TargetScan (http://www.targetscan.org/vert_72/) were used to search for the targets of miR-18a-3p (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to obtain the common targets from above mentioned databases. Heatmap software(ggplot2) was applied to cluster the enrichment terms obtained after Kyoto Encyclopedia of Genes and Genomes-based analysis of the common genes.The online databases miRwalk  was used to reverse transcribe RNA into complementary DNA (cDNA). Quantitative reverse-transcription PCR (qRT-PCR) was performed using an ABI 7500 sequence detection system  with SYBR Green Master Mix . The thermal conditions were as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. GAPDH was used as the internal reference of gene expression. Each sample was measured in triplicate. The U6 small RNA was used as the internal reference for estimating the levels of miR-18a-3p and other members in miR-17-92 cluster. All relevant PCR primers used in this study are listed in Trizol (Invitrogen) was used to extract total RNA from the muscle tissues according to the manufacturerNE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific) was used to extract nuclear-cytoplasmic fraction according to the manufacturer’s instructions. NP-40 lysis buffer  and phenylmethylsulfonyl fluoride  were used to obtain total protein. Protein quantification were performed by BCA Protein Assay kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. The available protein was denatured with 5× sodium dodecyl sulfate (SDS)-PAGE denatured protein loading buffer  at 98°C for 5 min. Next, SDS-polyacrylamide discontinuous gel electrophoresis was used to separate the denatured protein; then, the protein was transferred to nitrocellulose filter membranes  by wetting transfer method for 90 min. Immunoblotting was conducted using specific antibodies listed in Differentiated HSMM myotubes or primary myotubes were fixed in 95% ethanol. Then, 0.3% Triton X-100 was used for 15 min for cell membrane penetration. The samples were washed three times, for 5 minutes each time, and then blocked with goat serum for 2 h. The samples were then incubated with anti-HuR , anti-MyoG , and anti-myosin heavy chain  overnight at 4°C, and then with Alexa 488-conjugated or Alexa 555-conjugated secondary antibody (Abcam) for 30 min at 25°C. Next, 4′,6-diamidino-2-phenylindole  was used to stain the cell nuclei, which were observed under a fluorescence microscope . The degree of differentiation was measured by a fusion index, which is defined as the number of nuclei in the myotube and as a percentage of the total nuclei.t‐test was used for separating the means of the normal data, whereas Mann–Whitney U test for data that deviated from normal. Spearman’s rank-order correlation was used to test correlations of data with non-normal distribution. A p value < 0.05 was considered as statistically significant. SPSS 22.0  and GraphPad Prism 8.0  were used for all statistical analyses.Continuous data were expressed as means ± standard deviation for normal distribution or as median and interquartile range for those without normal distribution. Student’s The clinical characteristics of the patients with IMNM are presented in We found that the expression level of miR-18a-3p was significantly higher in patients with IMNM than in the HCs (p=0.0002) , we furtr = 0.493, p = 0.037; r = 0.576, p = 0.012); however, the muscle activity component of the Myositis Disease Activity Assessment Tool by VAS (MDAAT-muscle) was negatively correlated with miR-18a-3p expression   by binding to the mRNAs of inflammatory cytokines  , 32. We Overall, our study revealed that miR-18a-3p was significantly upregulated, whereas its target gene HuR was downregulated in the muscle tissues of patients with IMNM compared to DM and HCs. miR-18a-3p were negatively correlated with muscle activity in patients with IMNM, but the expression levels of HuR were positively correlated with disease activity in patients with IMNM. Finally, miR-18a-3p and HuR might regulate myoblast differentiation Figure 6The data presented in the study are available from the corresponding authors upon request.The studies involving human participants were reviewed and approved by Ethical Review Committee of the China–Japan Friendship Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LY, YZ, GW, and XS participated in the conception and design of the experiments. LY and YZ performed the experiments and data analysis. LY, YZ, and XS wrote the manuscript. FC, QP, XL, and GW supervised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Elite Medical Professionals project of China-Japan Friendship Hospital (NO.ZRJY2021-GG14), the Youth Program of the National Natural Science Foundation of China , Major Research Plan of the National Natural Science of China(grant number 91542121), National Natural Science Foundation of China(grant number 81971531),Capital Health Research and Development of Special Programs (grant number 2014–4-4062), and The Fundamental Research Funds for the Central Universities (grant number 3332020074).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Self-report screening instruments are frequently used as scalable methods to detect common mental disorders (CMDs), but their validity across cultural and linguistic groups is unclear. We summarized the diagnostic accuracy of brief questionnaires on symptoms of depression, anxiety and posttraumatic stress disorder (PTSD) among Arabic-speaking adults.Five databases were searched from inception to 22 January 2021 (PROSPERO: CRD42018070645). Studies were included when diagnostic accuracy of brief  psychological questionnaires was assessed in Arabic-speaking populations and the reference standard was a clinical interview. Data on sensitivity/specificity, area under the curve, and data to generate 2 × 2 tables at various thresholds were extracted. Meta-analysis was performed using the diagmeta package in R. Quality of studies was assessed with QUADAS-2.Nparticipants = 4042) reporting on 17 questionnaires with 5–25 items targeting depression/anxiety (n = 14), general distress (n = 2), and PTSD (n = 1) were included. Seventeen studies (53%) scored high risk on at least two QUADAS-2 domains. The meta-analysis identified an optimal threshold of 11  for the Edinburgh Postnatal Depression Scale (EPDS) , 7  for the Hospital Anxiety and Depression Scale (HADS) anxiety subscale and 6  for the depression subscale , and 8  for the Self-Reporting Questionnaire (SRQ-20) .Thirty-two studies (We present optimal thresholds to screen for perinatal depression with the EPDS, anxiety/depression with the HADS, and CMDs with the SRQ-20. More research on Arabic-language questionnaires, especially those targeting PTSD, is needed. A meta-analysis across 39 countries indicated a lifetime prevalence of 29.2%, although this estimate varies across subgroups , the Hospital Anxiety and Depression Scale (HADS), and the PTSD Checklist (PCL). Brief instruments can be useful for routine screening in primary and stepped care  to identify individuals with a CMD compared to individuals without a disorder is called et al., et al., et al., et al., et al., Previous systematic reviews on the validity of screening instruments have focused on a specific instrument  . We followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA-DTA) checklist  as index terms or free-text words: ‘Sensitivity and Specificity’, ‘Reference Standards’, ‘Diagnostic Self Evaluation’, ‘Common Mental Disorders’, and ‘Arabic speaking populations’. The full search strategy is attached as online Supplementary Appendix 2. We restricted the search to articles, proceeding papers, conference papers, and electronic collections. We also identified studies by screening literature lists of included studies . We did not base our definition on, e.g. ‘time of administration’, given that time to complete a measure might vary among groups and literacy levels. Reference standard – a diagnosis made through a structured clinical interview or by a clinician based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM) , and stress-related disorders. Anxiety disorders include generalized anxiety disorder (GAD), panic disorder, phobia, agoraphobia, or social anxiety disorder. PTSD and acute stress disorder are included (as anxiety disorders in DSM-IV or as trauma- and stress-related disorders in DSM-5). We excluded papers in which the diagnosis was based on a questionnaire, observation checklist, chart review, or self-reported diagnosis. We also excluded studies that did not provide data to calculate sensitivity/specificity.The full search yield was reviewed for inclusion by two independent reviewers (AdG/JU) on the basis of title and abstract. Both reviewers assessed full-texts of the remaining articles. Discrepancies were resolved by discussion, and remaining queries were discussed with a third reviewer (MS). The following inclusion criteria had to be met: Data were extracted independently from each study by two reviewers (AdG/IS) using a coding scheme , (2) whether data on interviewer variation (e.g. inter-rater reliability) for the (semi)structured interview fell within an acceptable range (domain 3), and (3) whether all participants received a reference standard (domain 4). See online Supplementary Appendix 3 for item specifications.Risk of bias was independently assessed by two reviewers (AdG/IS) using the quality assessment tool of diagnostic accuracy studies (QUADAS-2)  and type of outcome. For every study, we tabulated the questionnaire, reported cut-off scores and outcome measures. In this review, we present cut-off scores as rounded numbers (e.g. ‘5’), whereby individuals are considered positive cases if they have that score at minimum (e.g. 5 or above). Meta-analysis was performed when at least three studies with a comparable outcome for a specific questionnaire were included. Multiple thresholds were modelled for studies reporting a range of cut-off scores  were conducted in Arab countries. Participants , with mean age range 28–82 years, were selected from clinical settings , community settings , or both . Nine (28.1%) studies included only women, two (6.3%) only men, 17 (53.1%) mixed samples, and two (6.9%) did not report gender (6.9%). None of the questionnaires were locally developed, but all were translations of English-language instruments: in the majority of studies, questionnaires  were locally translated, five (15.6%) used/adapted already existing translations, and seven (21.9%) did not report on translation. In 20 studies (62.5%), questionnaires were administered by interviewers.One study was conducted among a sub-sample of Arabic-speaking migrants in Australia (Barnett n = 13) or lay-interviewer (n = 3); six studies did not report on the type of interviewer. In the other 10 studies (31.3%), a clinician diagnosis according to the DSM/ICD was made. El-Hachem et al. (Twenty-two studies (68.7%) used a (semi-)structured clinical interview as reference standard. Seven studies used the Mini International Neuropsychiatry Inventory (MINI), five the Structured Clinical Interview for DSM (SCID), four the Composite International Diagnostic Interview (CIDI), three the Clinical Interview Schedule (CIS), two the Present State Examination (PSE), and one the Diagnostic Interview Schedule (DIS). These (semi-)structured interviews were conducted by a clinician  to any anxiety disorder, with a sensitivity of 57% and a specificity of 53%, and one study compared the PHQ modules panic, with a sensitivity of 47% and a specificity of 96%, and GAD, with a sensitivity of 37% and a specificity of 96%, to corresponding DSM-IV criteria.We found three instruments targeting combined anxiety/depression that were compared to a diagnosis of anxiety and/or depression. The HADS was evaluated in four studies. The sensitivity of the anxiety subscale ranged from 62% to 85%, and its specificity from 62% to 91%. The sensitivity range of the depression subscale was 54–90%, and specificity range 70–99%. One study evaluated the HSCL-25. The sensitivity of the anxiety subscale was 84%, and its specificity 59%. The sensitivity of the depression subscale was 82%, and its specificity 70%. The Primary Care Anxiety and Depression scale was evaluated in one study, which found a sensitivity of 82% and a specificity of 77%.We found one instrument targeting PTSD symptoms. The Screen for Posttraumatic Stress Symptoms (SPTSS) had a sensitivity of 89% and a specificity of 89% compared to a PTSD diagnosis.Lastly, we identified two general distress instruments that were compared to a diagnosis of any CMD. The 20-item Self-Reporting Questionnaire (SRQ-20) was investigated in six studies, of which one study also included a psychosis item. The sensitivity range was 71–100%; the specificity range 70–95%. The 12-item General Health Questionnaire (GHQ-12) was evaluated in one study and had a sensitivity of 83% and a specificity of 80%.Online Supplementary Appendix 5 presents a visual representation for all instruments for which we included at least three studies.The QUADAS-2 results are evaluated at item-level and do not incorporate an overall quality score . Eleven Patient Selection was low in the majority of studies. Studies scored high risk if a case-control design was used  reporting on the same questionnaire and comparable target condition. Optimal thresholds for the EPDS, HADS anxiety and depression subscales (HADS-A and HADS-D), and SRQ-20 could be estimated. Two studies on the SRQ-20 were excluded from meta-analysis, because of missing data to calculate the 2 × 2 table ; resulting in a practically relevant optimal cut-off score of 11, with a pooled sensitivity of 76.9%  and a specificity of 85.2% (95% CI 78.4–90.1).Our model identified 11.08 as optimal threshold for the EPDS (n = 4) identified 7.17 as an optimal threshold, indicating a practically relevant cut-off score of 7 with a pooled sensitivity of 71.9% (95% CI 41.9–90.1) and a specificity of 78.5% (95% CI 67.3–86.6). The HADS-D model (n = 4) identified 5.97 as an optimal threshold, with 6 as the closest, practically relevant cut-off score, having a pooled sensitivity of 73.0% (95% CI 48.9–88.4) and a specificity of 88.6% (95% CI 75.7–95.1). CIs for the sensitivity/specificity estimates of the HADS subscales were wide, also illustrated by widely varying ROC curves in The HADS-A model (n = 4) identified 8.36 as an optimal threshold, indicating a practically relevant cut-off score of 8 with a pooled sensitivity of 86.0% (95% CI 78.0–91.4) and a specificity of 83.9% (95% CI 58.1–95.1). The questionnaire's CIs associated with the pooled specificity were particularly wide.Finally, the SRQ-20 model , population (e.g. pregnant women v. elderly), and setting . Although this review focused on Arabic-speaking populations, the global Arabic-speaking community cannot be considered as one monolithic cultural group with identical idioms of distress or manifestations of psychological distress /specificity (85.2%). This threshold is lower compared to the original cut-off of 13 in English-speaking populations  and specificity (78.5%), and of 6 for the HADS-D (sensitivity: 73.0%/specificity: 88.6%). CIs for HADS were wide, indicating uncertainty about the estimated psychometric properties. In a recent IPDMA on the accuracy of the HADS-D to estimate depression prevalence, Brehaut et al.  found th . In other words, 14% of individuals with a disorder will remain undetected, while 16.1% of individuals without a disorder screen positive. The CIs for specificity were relatively wide. We therefore suggest that the SRQ-20 cut-off of 8 is useful for screening purposes to v. screening in stepped care).This review has several strengths and limitations. A strength is that it provides researchers and clinicians working with Arabic-speaking populations with an overview of the validity of brief screening tools, and empirically grounded recommendations for thresholds. We provided the results of multi-threshold models, rather than bivariate models in which only one threshold per study can be pooled. In doing so, we were able to provide the pooled accuracy statistics at different cut-off scores, allowing researchers and clinicians to decide which threshold is most suitable structured interviews and (unstructured) clinician diagnoses. Clinician diagnoses may be less reliable than (semi-)structured interviews (Segal and Williams, The clinical implications of this review are that a cut-off of 11 on the Arabic-language EPDS could be used as a screener for depression in perinatal populations to optimize a balance between sensitivity/specificity. For ruling out the presence of any CMD with the SRQ-20, we recommend using a cut-off score of 8. The evidence for the HADS to screen for depression and/or anxiety was not convincing as results were substantially heterogeneous.This review also stresses the paucity of evidence on anxiety and PTSD screeners. Future studies are needed to investigate the diagnostic accuracy of questionnaires to detect anxiety and PTSD in Arabic-speaking populations given the amount of Arabic-speaking refugees at risk for developing stress-related disorders (Peconga and Høgh Thøgersen, This review identified 17 brief questionnaires in the Arabic language that were investigated on diagnostic performance, with limited availability of evidence for PTSD instruments. The meta-analysis provided optimal cut-off scores for the EPDS, HADS, and SRQ-20.


Furthermore, FTIR revealed the basic vibrational signatures at ~3300 cm−1, 1023 cm−1, 1400 cm−1 for O-H, C-O, and C-H groups, respectively, for the carbon and oxygen atoms of the C60 molecule. NMR studies exhibited the different footprints and magnetic moments at ~7.285 ppm, explaining the unique underlying electrochemical attributes of the fullerene molecule. Such unique electronic and physico-chemical properties of the caged carbon structure raise hope for applications into the drug delivery domain. The in-vitro dose-dependent application of C60 elicits a toxic response against both the breast adenocarcinoma cell lines and pathogenic microbes. That enables the use of AgNPs decorated C60 endo fullerene molecules to design an effective anti-cancerous drug delivery and antimicrobial agent in the future, bringing a revolutionary change in the perspective of a treatment regime.At present, the potential role of the AgNPs/endo-fullerene molecule metal nano-composite has been evaluated over the biosystems in-vitro. The intra-atomic configuration of the fullerene molecule (C The electrons in their structures are restricted to moving in all three directions along the axes of x, y, and z, respectively. The degree of freedom of electrons dictates the movement of underlying electrons in the structures that govern the myriad differential applications from electronics to the bio-medical field . The basal medium DMEM enriched with FBS (10%) and streptomycin sulfate andbenzylpenicillin antibiotics at a final concentration of 100 µg/mL and 100 U/mL were used for all cell culture experiments. The cells were incubated at 37 °C and 5% CO2 during the experiments. Later, the cells were treated with Trypsin-EDTA (0.25%) at ~75–80% assemblage and implanted in 96-well plates at a density of 5 × 103 cells/well for the 3--2,5-diphenyltetrazolium bromide  colorimetric assay [The cytotoxicity assay of C, MDA-MB-31. The bic assay .60-AgNPs nano-composites [Cytotoxicity of the cell lines was determined using MTT assay at both 24 and 48 h of incubation with the Cmposites . The drut-test (p ≤ 0.05).All the assays were carried out in triplicates to ensure the reawakening of the results. The antimicrobial and antioxidant assays results were depicted as a percentage of inhibition, while the cytotoxicity results were depicted as percent viability, comparative to the control. The values gleaned in the assays for the various treatment groups vs. the controls were statistically determined using a Student’s 60-AgNPs, as shown in 60 get shifted to form partial shoulder vibrational signatures at C-O indicating the formation of the nano-composites [−1, 1023 cm−1, 1400 cm−1 are indications for the presence of functional groups for O-H vibrations, C-O and C-H stretching vibrations, respectively [The intrinsic functional groups and the associated vibrational spectroscopic evidence highlight the observations obtained for the nano-composites of Cmposites . The vibectively . The dif−1, 1023 cm−1, and 1400 cm−1. The pristine AgNPs from the previous studies have highlighted the incidence of the vibrations of the functional groups at ~2363 cm−1, 1625 cm−1, and 1126 cm−1, which are the predominant functional stretching vibrational groups in the pristine AgNPs [60 framework usually shows stretching vibrations for their native carbon structure at ~576 cm−1, 1097 cm−1, 1181, and 1427 cm−1 wavenumbers, respectively [60 at 576 cm−1 is associated with a primarily radial motion of the carbon atoms, while the other bands at 1181 and 1427 cm−1 are associated with a tangential motion of carbon atoms. The most characteristic vibrational mode is the pentagonal ‘pinch’ mode at 1427 cm−1 [60 are clearly seen to interact among them, resulting in a blend and mixture in their relevant coordination chemistry resulting in the production of shifted and mixed wavenumbers as mentioned hitherto in the section. Such observations correctly justify the formation of the wet procedure mediated synthesis of nano-composites of two different entities of metallic clubbed to the organic framework. The resulting formation of the metal–carbonaceous composites would help explore the intrinsic electronic and electrochemical significance of both the entities in the same platform, which is otherwise elicited independently as studies previously by several authors in biological and electronic applications. The resulting interactions between the metallic AgNPs and the organic framework of C60 make the vibrational shifts as mentioned hitherto for the C-O, C-H stretching numbers at ~3300 cmne AgNPs . Pristinectively . The wav427 cm−1 . Such sp60 molecule. The composite helps in the formation of mixed magnetic alignments in the nanoscaled system. The formation of the nano-composites indicates the formation of interlinked nano-composite architecture, indicating the formation of lattice fringes in the two independent systems. The magnetic moments of the two associated systems are indicated in the form of ppm values, as seen from the figure. It could be understood from the existing NMR spectra that were owing to the induction of surface electrons towards the basic structure of the buckyball fullerenes (C60), the intermixing redox state is producing a native magnetic moment pronounced at ~7.285 ppm against the control peak at~1.577 ppm, respectively [It could be seen from the NMR plot in ectively  in the c2 carbon skeleton of C60 molecules. The standard peak at ~1.577 ppm indicates the control peak of 13C NMR taken during measurements for comparison. It is understood from the observations that were owing to the predominant peaks at ~1.577 ppm and ~7.7 ppm, the structural entities of the outer electron level from both the interacting elements gets shared in their co-mixed architecture. The structural entanglement of the interacting elements in the nano-composites uncovers the principle of redox state interactions and exchanges, which is assumed to be of π–π interactions between the organic C60 molecule and the metallic AgNPs, respectively. It is further assumed that due to the presence and predominance of surface plasmonic electrons [The scanning range of the NMR spectroscopy has been selected at 1–8 ppm, whereby ~7.5 ppm there is an observation of the peak intensity, indicating the formation of magnetic moment characterization for the as-prepared nano-composites, which comprises the predominantly architecture of splectrons  over thelectrons .60-AgNPs nano-composite topographically. The surface topography and surface texture can be easily understood from 60 buckyball structure (indicated by agglomerated particles) could be seen in the figure. The image could be corroborated with the respective EDAX analyses, whereby the incidence of a Silver (Ag) peak besides Carbon (C) peak indicates the formation of the nano-composite structures of C60-AgNPs.It could be seen from 60) molecule [60 molecule will aid in the overall charge augmentations taking place over the C60 molecule, as evident from the nano-composite formations [60-AgNPs nano-composite at the nanoscale would enable different biomedical applications from carrying desired drug payloads to myriad bioelectronic applications.It could be clearly understood that owing to such elemental depositions of AgNPs over the buckyball architecture . The entire C60-AgNPs nano-composites form an electronically mediated system harboring electrostatic charges contributed from the pristine C60 molecules and the positive surface defect-induced AgNPs structures. The surface electrons existing over the AgNPs migrate from its surface to the surface of C60 molecules, derived from the charge–charge interactions and surface energy between the metallic-carbon structures, thereby achieving a thermodynamically stable system. Such minimized entropy system of the nano-composites aids in employing the system in different bioelectronic applications.As indicated from 60-AgNPs nano-composites in an aqueous solution were calculated using DLS . The average size was calculated to be 68.27 ± 0.489 nm . It is assumed that, albeit the interaction mechanism involved in the formation of the metallic and organic framework of composites in the solvent of distilled water, we ascertain the net average diameter of the composite to be the same as calculated and hence mentioned. The dynamics and kinetics of the as-synthesized nano-composites in the solution are beyond the scope of the present study and would be further analyzed vertically in solution kinetical studies in the future, which would aid to uncover the actual size distribution pattern of the individual nano-composites when compared to its participating individual components. The pristine AgNPs were calculated from the solution phase interaction of the average size distribution to ~15–20 nm and ~50 nm for the C60 molecules, which is in close agreement with previous studies [It could be seen from the figure of DLS and its corresponding zeta analyses that owing to the bottom-up approach of the synthesis of nano-composites of metal-organic framework, the polydispersity, and the granulometry analyses have been strictly analyzed in the present study. The rough edges of the metallic AgNPs and the symmetric Curements a that up studies .Similarly, from the Zeta-sizer analyses, the composite suspension indicates the stability of the as-prepared nano-composites solution. We have achieved a zeta value ~ −26.6 ± 4.29 mV, indicating sparingly stable composite formation. It is understood that the more negative value of the nanosystems indicates the crucial role played by the involved capping agents or associated agents in making the nanoscaled system away from the Reddick threshold . As envi60-AgNPs nano-composites was dose-dependent  is one of the unique and versatile carbon-based nanomaterials valued in biomedicine development. It acquires remarkable antioxidative capacity, which has fashioned it a strong and major element to be utilized in skin-related cosmetics and anti-aging products . Besidesependent . The scaradicals .C60 is toperties . Since ooperties ,48,49. E60-AgNPs nano-composite was preliminarily determined by agar–well diffusion method against seven human pathogenic bacteria Vibrio cholerae, Shigella dysenteriae, Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus licheniformis, and fungal strain Candida albicans as shown in 60-AgNPs nano-composite has the highest activity against S. dysenteriae. Further, the antimicrobial assay was carried out by using the micro broth dilution method to corroborate the results found in the agar–well diffusion method. S. dysenteriae among all the selected bacterial strains exhibited an intensified inhibition % by C60-AgNPs nano-composite.The antimicrobial activity of C50 calculations display an evidenced antimicrobial activity of V. cholera (105.44 ± 3.67 µg/mL), S. dysenteriae (52.72 ± 0.27 µg/mL), S. typhimurium (90.10 ± 0.35 µg/mL), E. coli (79.54 ± 0.51 µg/mL), S. aureus (91.15 ± 0.77 µg/mL), S. epidermidis (84.97 ± 0.68 µg/mL), B. licheniformis(81.99 ± 1.37 µg/mL), C. albicans (76.61 ± 1.98 µg/mL) which has been backed and substantiated with several previous research findings in the similar lines. When Ag NPs and C60 were tested separately, Ag NPs showed superior antimicrobial activity than C60. Interestingly, the nano-composite C60-AgNPs showed synergistic antimicrobial activities. C60-AgNPs nano-composite showed significantly higher antimicrobial activity in comparison to Ag-NPs and C60 against all eight tested microorganisms.In contrast, five pathogenic bacterial strains exhibited a more than 90% inhibition rate, and the pathogenic fungus also exhibited >90% inhibition. Among the MICs studies, as shown in 60-AgNPs nano-composites are highly effective against S. dysenteriae. Moreover, the as-prepared nanomaterial is also equally potent against other pathogens, which signifies the IC50 values. The IC50 < 100 µg/mL of the current tested microorganisms endorsed the potential applications of C60-AgNPs nano-composite materials as an antimicrobial candidate and as a supporting engagement in developing biomedical devices and other drugs packaging materials. Fungal infections are prevalent and frequently observed in the long-term storage of any stuff, and the anti-candidal activity of such nanomaterials makes it an emerging antifungal nanomaterial that can be used in different bio application fields. There are many reports on the potential antimicrobial activities of both pristine C60 and AgNPs. The present findings of enhanced antimicrobial results of these nano-composites make promising hope of profound applications of unique combinatorial nanomaterials organo-metallic in nature. Fullerene has been studied well in potential applications in different cosmetics and other skincare industries due to its hypothesized strong antioxidant activity [60-AgNPs nano-composite.From the MICs value, it was also proved that Cactivity . Hence n60-AgNPs nano-composite may be associated with some well-described mechanisms such as the adhesive capability of C60-AgNPs onto the cover of cell wall and membrane, prejudicial activity on major intracellular structures  and important biomolecules  due to penetration capability due to nano size, induction of oxidative stress and cellular toxicity due to reactive oxygen species (ROS) generation and harmful free radicals and inflection of signal transduction pathways [60-AgNPs nano-composite also inflects the human immune system by coordinating inflammatory response [The promising antimicrobial actions of Cpathways . Besidesresponse , which f60-AgNPs nano-composite in the culture at a varied concentration (0.97 nM to 1000 nM) for 24 h at 37 °C. The MTT assay was adopted to determine human keratinocyte (HaCaT) cell viability upon treatment with C60-AgNPs nano-composite  was accessed in a similar manner. In contrast to results with normal cells (HaCaT), the cytotoxicity assay of C60-AgNPs nano-composite with MDA-MB-231 revealed axiomatically higher levels of cytotoxicity (60-AgNPs nano-composite for 24 h lowered the cell viability to ~20.46 %. A time and dose-dependent cytotoxicity of C60-AgNPs nano-composite against MDA-MB-231 cells has been reported earlier [The cell viability of Ctoxicity . The int earlier ,56,57.60-AgNPs was raised. The percentage of viable cells was 62.86% and 99.29% after interaction with 250 nM and 0.97 nM C60-AgNPs solutions, reciprocally. The present results proved that MDA-MB-231cells displayed a dose-dependent response in cytotoxic activity study. However, the cytotoxicity did not occur to be time-dependent as a very minute difference in the percentage of viability was observed after both 24 and 48 h of incubation, maintaining the similar concentration of C60-AgNPs (60-AgNPs in breast adenocarcinoma cells [60-AgNPs nanomaterials are brought into a cell and disarray the whole metabolism [50 values are lower in MDA-MB-231 cells than HaCaT cells after 24 h and 48 h of susceptivity. Moreover, the IC50 values for the C60-AgNPs treatment were lower in MDA-MB-231 cells than in HaCaT cells after both 24 h and 48 h of susceptivity (50 values of C60-AgNPs compared to doxorubicin strongly proved to be the usage in cancer therapy in biomedical sectors.The percentage of viable MDA-MB-231 cells was reduced as the dose of C60-AgNPs . The curma cells . The crutabolism . During eptivity . The IC560 nano-composites. Diverse spectroscopic and micro graphical techniques have been used to uncover the electronical and chemical behavior of the synthesized nano-composites, possessing organic carbon-based entities as well as their metallic counterparts in its native structure. Moreover, when the nano-composite was assorted with cell lines of MDA-MB-231, it elicited a meagre cytotoxicity behavior. Such a unique combination of zero-dimensional carbon framework systems possessing buckyball structure, ingrained with a spherical nano-particulate system having huge surface plasmonic resonance, surface energy, and surface area attributes. Thus, this would be a novel approach to mitigating several bioelectronics issues to biomedical applications.The study helps to explore the various physico-chemical features and bioactivities of the metallic AgNPs and C


The relationship between skeletal muscle mass at the beginning of the post-acute rehabilitation phase and rehabilitation outcomes has been scarcely investigated. The aim of this study was to investigate the impact of the existence of sarcopenia upon admission to a post-acute COVID-19 patient rehabilitation unit on body composition and functional and respiratory capacity at discharge. Thirty-four post-acute COVID-19 patients were referred to our Rehabilitation Unit from different COVID Hospitals in northern Italy. Body weight loss, body composition, handgrip strength, functional parameters, oxygen saturation and related perception of dyspnea in several positions were measured before and after a 28-day multidisciplinary rehabilitation program. Spirometry was performed only upon admission. The intervention included psychiatric support, cognitive behavioral therapy, nutritional therapy and physiotherapy, including aerobic and resistance training. Training volume was 45 min/session, 6 sessions/week. Upon admission, the prevalence of sarcopenia among our patients was 58%. In all of the 34 patients, we observed a trend of improvement in all of the respiratory, body composition, muscle strength and functional parameters considered. Monitoring muscle mass and strength in post-acute COVID-19 patients appears to be a key predictor of rehabilitation outcomes. Early diagnosis of sarcopenia therefore appears to be of paramount importance in the management of post-acute COVID-19 patients. COVID-19 is an infectious disease caused by SARS-CoV-2, leading to a severe acute respiratory syndrome and multi-organ failure . In the From April to June 2020, we recruited 34 post-acute COVID-19 patients re-referred to our Rehabilitation Unit from several COVID hospitals in the north of Italy, consecutively. All patients presented a history of COVID-19 disease documented by real-time PCR and next-generation sequencing positive swabs, and with different clinical manifestations. Before admission to our Rehabilitation Unit, patients had to present two consecutive negative swabs for SARS-CoV-2. This study was conducted according to the Declaration of Helsinki of the World Medical Association and was approved by the Ethics Committee of the Istituto Auxologico Italiano (2020_05_19_04). Written informed consent was obtained from all experimental patients.2) was calculated. Upon admission, individual weight loss was calculated from the body weight value prior to the COVID-19 disease, held as “habitual body weight”. Body composition was assessed in the morning in a comfortable room at a temperature of 22–25 °C by Bioelectrical Impedance Analysis  with the patient in a supine position, with the lower limbs slightly separated and an empty bladder. Whole-body resistance (Rz) and reactance (Xc) were measured by expert operators with an impedance analyzer that delivered a 50 kHz sinusoidal alternating current connected to four surface electrodes placed on the right foot and hand [2 [Body height (to the nearest 0.1 cm) and weight (to the nearest 0.1 kg) were measured in light indoor clothing. Body mass index , our multi-disciplinary rehabilitation program is intended to help recover from functional impairment and limitations of the respiratory, cardiac, musculoskeletal and neurological systems. It includes psychiatric support, cognitive behavioral therapy to reduce the post-traumatic stress symptoms and nutritional treatment to improve malnutrition. All patients recruited were from other hospitals in the same or neighboring regions. Based on the medical records, 11 of the 34 patients were hospitalized in the ICU. Unfortunately, we are not aware of the number of days spent in the ICU because they were not always reported. In our sample, prevalence was 17.1% for hypertension, 16.4% for cardiac-cerebrovascular disease and 9.7% for diabetes. Length of the program was on average 28 days. Each patient was individually trained by a physical therapist to initially perform sitting-to-bed exercises for upper-body conditioning , our mul and peri2 or usual body weight for BMI < 28 kg/m2 before COVID-19 disease. Likewise, we calculated protein intake in the range of 1 to 1.3 g/die/kg body weight. An ideal energy ratio of fat to carbohydrate was 30:70, or 50:50 in patients supplemented with O2. In an additional way, as recommended by ESPEN guidelines, patients were supplemented with oral multi-minerals  and multivitamins  ; Italy), and vitamin D  and essential amino acids (4 to 8 g/day).The nutritional intervention was based on the recent practical guidelines established by the European Society for Clinical Nutrition and Metabolism (ESPEN) for the nutritional management of COVID-19 patients [Clinical examination, anthropometry, laboratory findings, body composition (ASM and PhA), hand grip strength (HG), performance test (TUG), oxygen saturation and related perception of dyspnea in the lying, sitting and standing positions and after completion of TUG were performed at T0 and T1 to evaluate the effectiveness of the rehabilitation program. Spirometric testing was performed only at T0 to evaluate the baseline pulmonary capacity of patients.p-value < 0.05 was considered to be statistically significant. Statistical analyses were performed with SPSS 25.0 .Continuous variables were expressed as median and interquartile range. Differences between groups were assessed using Fisher’s test for continuous variables, or the c2 test for categorical data, as appropriate. A 2) were classified as sarcopenic. The number of patients admitted to the intensive care unit in the previous hospitalization was 8/20 based on the clinical records. Fourteen patients  years, mean BMI 28.4 (8.9) kg/m2) were not sarcopenic. The number of patients admitted to the intensive care unit in the previous hospitalization was 3/14.Twenty patients  years, mean BMI 21.0 (4.2) kg/mIn our sample, comorbidity prevalence was 17.1% for hypertension, 16.4% for cardiac-cerebrovascular disease and 9.7% for diabetes. Anthropometric, body composition, muscle strength and functional values of both subgroups at admission (T0) and discharge (T1) are reported in 2 after TUG showed a statistically significant improvement at T1. Spirometry was evaluated only at T0: we found a FEV1 of 1.98 L (SD: 0.70 L) and a FVC of 2.30 L (SD: 0.81 L) in S, and a FEV1 of 2.16 L (SD: 0.73 L) and an FVC of 2.58 L (SD: 0.88 L) in NS. In S, the correlation between ASM and FVC and FEV1 was statistically significant , whereas in NS, the correlation was not significant. Main laboratory findings at T0 and T1 are reported in Respiratory parameters in S and NS at T0 and T1 are reported in p = 0.001) and NS (p = 0.009) groups showed a statistically significant improvement only in female patients. HG improved in S patients, but the improvement was only significant in females (p = 0.035 *). TUG improved significantly in both S (p = 0.039 *) and NS (p = 0.045 *) groups. A predominance of different muscle fibers and a different cross-sectional area could account for gender differences, as type 1 fibers are predominant in females. The improvement in ASM observed only in female S patients may be attributable to such gender differences. Increased muscle mass may require more dietary protein in order to achieve a positive net protein balance and overcome muscle protein breakdown [Upon admission to the Rehabilitation Unit after the acute phase, we documented a 58% prevalence of sarcopenia among our 34 post-COVID-19 patients, according to the cut-off values by Cruz-Jentoff et al. This is in line with recent reports on the prevalence of sarcopenia in intensive care units . Known freakdown . It shoureakdown . Additios p = 0.0 *. TUG ireakdown . It can reakdown . Muscle reakdown . Based oWe documented a 58% prevalence of sarcopenia among our 34 post-COVID-19 patients. This sample is small and further investigation is necessary. Early diagnosis of sarcopenia appeared to be of paramount importance in the management of post-COVID-19 patients to improve their rehabilitation outcomes and long-term prognosis [


Deep sequencing could improve understanding of HIV treatment failure and viral population dynamics. However, this tool is often inaccessible in low- and middle-income countries.To determine the genetic patterns of resistance emerging in West African HIV-1 subtypes during first-line virological failure, and the implications for future antiretroviral options.Participants were selected from a Nigerian cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy. Whole HIV-1 genome sequences were generated from first-line virological failure samples with Illumina MiSeq. Mutations detected at ≥2% frequency were analysed and compared by subtype.P = 0.002), and ≥3 TAMs were more common in G than CRF02_AG . Subtype G viruses also contained more RT cleavage site mutations. Cross-resistance to at least one of the newer NNRTIs, doravirine, etravirine or rilpivirine, was predicted in 81% of participants.HIV-1 sequences were obtained from 101 participants . Thymidine analogue mutations (TAMs) were detected in 61%, other core NRTI mutations in 92% and NNRTI mutations in 99%. Minority variants (<20% frequency) comprised 18% of all mutations. K65R was more prevalent in CRF02_AG than G subtypes (33% versus 7%; Extensive drug resistance had accumulated in people with West African HIV-1 subtypes, prior to second-line ART. Deep sequencing significantly increased the detection of resistance-associated mutations. Caution should be used if considering newer-generation NNRTI agents in this setting. First-line regimens comprising two NRTIs and an NNRTI can be very successful, with one meta-analysis reporting viral suppression in over 80% of people retained in care for up to 5 years.pol gene Sanger sequencing technology.,,Most of the literature on HIV drug resistance is derived from partial Other parts of the genome that are not usually studied, such as the HIV-1 cleavage sites, may also affect the response to therapy.,The greatest HIV-1 genetic diversity is seen in West and Central Africa, a region with multiple circulating clades such as subtype G, CRF02_AG and CRF06_cpx.Participants were selected from a cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy at the University of Abuja Teaching Hospital, Nigeria. Eligible participants were identified by searching the IHVN database and laboratory records. People aged >15 years were included if they had experienced virological failure during first-line NNRTI-based ART  and had a stored plasma sample available from this timepoint.XT automated target enrichment protocol  with in-house HIV baits.Nucleic acid was extracted using the QIAamp Viral RNA Mini Kit, QIAGEN . The cDNA was synthesized with SuperScript IV reverse transcriptase  and NEBNext second strand cDNA synthesis . Sample libraries were prepared as per the SureSelectK03455).HIV subtypes were determined by the REGA subtyping tool version 3.0.Ethical approval was granted by the IHVN Institutional Review Board, the National Health Research Ethics Committee of Nigeria, the University of Maryland, Baltimore Institutional Review Board and the University College London Research Ethics Committee (14865/002).Full-length HIV-1 genome sequences were generated from plasma samples from 101 participants. The participant characteristics are shown in A total of 616 resistance-associated mutations were detected in the 101 samples from all participants. Minority variants, present at 2%–20% frequency in the intrahost viral population, accounted for 18.2% (112/616) of all mutations. This included 49 (8.0%) present at 2%–5% frequency and 63 (10.2%) present at 5%–20% frequency. A further 54 mutations (8.8%) were present at 20%–50% frequency, 96 (15.6%) at 50%–90% frequency and over half  were present at ≥90% frequency within the viral population. There were 66 mutations that were not included in the analysis as they were detected at <2% frequency and therefore could not be distinguished from low-level sequencing errors.Overall, 59% of participants had minority variants. This included thymidine analogue mutation (TAM), core NRTI and NNRTI minority variants, which were detected in 28% (28/101), 19% (19/101) and 35% (35/101), respectively. Minority variants were usually detected along with other higher frequency mutations of the same class . HoweverThe most common individual mutations are shown in There was also a high prevalence of mutations among the 12 participants who were sampled less than 1 year after initiating first-line treatment: 6 (50%) had TAMs present at ≥2% frequency; 10 (83%) had core NRTI mutations ; and 12 (100%) had NNRTI mutations.P = 0.002). Three of the four CRF06_cpx samples also had the K65R mutation. There were no significant subtype differences in NRTI exposure: 48% (26/54) of participants with CRF02_AG had received tenofovir during first-line ART, compared with 38% (16/42) of participants with subtype G (P = 0.33). At least one of tenofovir or stavudine had been given to 72% (39/54) of participants with CRF02_AG and to 69% (29/42) with subtype G (P = 0.73).The K65R mutation was detected in the samples of 24% of participants (24/101), of whom 83% (20/24) had received tenofovir and 13% (3/24) had received stavudine, with two people having received both agents. All participants with K65R also had M184I/V. One-third of people with the CRF02_AG clade had K65R , compared with only 7% of people with subtype G , which is coded for by AAA or AAG, and arginine (R), which is coded for by AGA, AGG, CGT, CGC, CGA or CGG . All K65P = 0.004). There was a history of zidovudine exposure in 81% (34/42) of people with subtype G and 70% (38/54) with CRF02_AG (P = 0.24). Stavudine had been given to 41% (17/42) of people with subtype G and 30% (16/54) with CRF02_AG (P = 0.27). At least one of zidovudine or stavudine had been given to 88% (37/42) with subtype G and 78% (42/54) with CRF02_AG (P = 0.19). The ART duration did not differ significantly between participants with subtype G  and CRF02_AG .People with subtype G viruses were more likely to have three or more TAMs  compared with those with CRF02_AG . A substitution in the adjacent codon, K476Q, was noted in five sequences and accompanied F440Y in three of those. Other changes in the p51-p66 cleavage site were G436K in one sequence, A437V in seven, A437T in one and E438D in three. The protease-p51 cleavage site (CTLNF/PISPI) contained mutations in nine (9%) sequences: two I851V, six P853H/S/T and two I854V. The p66-integrase cleavage site (IRKVL/FLDGI) had mutations in 17 (17%) sequences: 2 I1405V, 10 K1407R and 5 V1408I. There were no significant subtype differences observed at the protease-p51 or p66-integrase cleavage sites.RT cleavage site mutations were found in 62% (63/101). Half (50/101) had changes in the p51-p66 cleavage site compared with the HXB2 reference amino acid sequence (GAETF/YVDGA). A phenylalanine-to-tyrosine substitution (F440Y) at the fifth codon of the cleavage site was present in 43% (43/101) of the study sequences and was more common in subtype G than CRF02_AG, at 88% and 11%, respectively (The predicted susceptibility to NRTI agents is shown in P = 0.37).All first-line virological failure sequences were predicted to be susceptible to the PIs lopinavir and atazanavir, which were prescribed for second-line therapy. However, only 11% of participants were prescribed two ‘active’ NRTI agents in their second-line regimen, to which the virus was predicted to be either susceptible or only low-level resistant. In 47% of participants, only one NRTI in their second-line regimen was predicted to be active, and in 43% no NRTIs were predicted to be active. The number of active agents in the second-line regimen did not differ significantly between participants who went on to have confirmed viral suppression and those who had virological failure during second-line ART. Of the 43 participants who received no active NRTIs, 23 (53%) had viral suppression and 20 (47%) had virological failure; of the 47 participants with one active NRTI, 19 (40%) had viral suppression and 28 (60%) had virological failure, and of the 11 participants with two active NRTIs, 4 (36%) had viral suppression and 7 (64%) had virological failure (Regardless of the mutation frequency threshold used, the Stanford algorithm predicted intermediate- to high-level resistance to nevirapine and efavirenz in >98% of participants. There was considerable cross-resistance to newer-generation NNRTI agents, to which these participants had not been exposed. Intermediate- to high-level resistance to at least one of doravirine, etravirine or rilpivirine was predicted in 81% of participants and 51% were predicted to have resistance to all three. Individually, intermediate- to high-level resistance to doravirine was predicted in 62%, to etravirine in 68% and to rilpivirine in 73%. A further 16% were predicted to have low-level resistance to doravirine, 5% to etravirine and 9% to rilpivirine.First-line virological failure was associated with extensive multiclass drug resistance prior to second-line ART switch in this cohort. Deep sequencing showed that around one fifth of RT mutations were minority variants, which would not have been detected by standard resistance testing methods. The detection of highly resistant viruses less than 1 year into therapy provides further evidence that routine viral load monitoring and adherence support are likely to be crucial from the outset of treatment to preserve first-line and second-line agents and to prevent the emergence of resistance.gag and pol sequences in GenBank.,The K65R mutation was significantly more prevalent in CRF02_AG viruses than subtype G, which was not completely explained by differences in tenofovir exposure or treatment duration. Nor was there a difference in the codon usage at position 65 or 64 that would be expected to predispose to a lysine-to-arginine substitution, such as the run of five adenosines before the second position of codon 65, which increases the likelihood of a mutation in subtype C viruses.,Most emergent resistance was already fixed in the viral population by the time of sampling, with over half of the mutations present at ≥90% frequency. Despite this, there were still many minority variants. These could represent emerging or waning resistance, or may reflect fluctuations in the frequency of mutations under changing drug pressure. Smaller studies in South Africa and Uganda also showed improved detection of drug resistance at first-line failure using deep sequencing technology.Lowering the mutation frequency threshold for predicting ART susceptibility resulted in more viruses being considered resistant to tenofovir and abacavir. Over two-thirds of participants had viruses predicted to be resistant to tenofovir, which is concerning as this agent is often used for second-line or third-line regimens, therefore subsequent lines of therapy could be jeopardized. Worryingly, without the benefit of genotypic resistance testing at the time of first-line virological failure, only 11% of participants were switched to second-line regimens with an NRTI backbone that was predicted to be fully active. Even if resistance testing had been available to influence the choice of second-line regimen, more than one-quarter of participants had viral genotypes that were predicted to be resistant to all available NRTI agents.However, the NRTI resistance detected at first-line treatment failure did not appear to correlate with subsequent second-line virological response. It may be that second-line treatment success is more likely to be influenced by host factors, such as adherence. This is consistent with the finding from the EARNEST trial of second-line HIV therapy, that people with no active NRTIs in their second-line regimen did not appear to have worse virological outcomes.Another issue, which may influence tenofovir susceptibility in particular, is the serendipitous effect that can occur when combinations of mutations cause lower levels of resistance than they generate individually. This is highlighted by a phenotypic analysis that showed the cytosine analogue mutation M184V mitigates against the effects of K65R, and so the overall impact on tenofovir susceptibility may not be significant when these two mutations coexist.An important finding was that two-thirds of participants had cross-resistance to newer-generation NNRTIs, some of which have been proposed even for people with a history of virological failure on first-generation NNRTI agents.,,,,,,,One limitation of this study is that the first-line resistance data were from people who subsequently switched to second-line ART, and therefore may not be representative of those who fail first-line ART but do not switch to second-line ART. For example, an analysis from the same study site found that one-fifth of Sanger sequences from 198 people who had failed first-line ART had no detectable drug resistance mutations.In summary, diverse West African HIV clades exhibit multiclass drug resistance with subtype variation at first-line virological failure, prior to second-line ART. The predominance of high-frequency mutations suggests that emergent resistance had become fixed in the viral population by the time of sampling. NRTI resistance was not associated with the virological response to second-line PI-containing regimens in this cohort. However, the NNRTI resistance profile suggests that newer-generation NNRTI agents are unlikely to be reliable in this setting.